Application of CRISPR/Cas9 genome editing technology for inhibition of hepatitis B virus replication

Abstract

Objective To evaluate the practicability of using CRISPR/Cas9 genome editing technology for inhibition of hepatitis B virus (HBV) replication. Methods Two sgRNA targeting sites were designed for the S region of HBV genome. The CRISPR/Cas9 expression plasmids specific for HBV were constructed and then transfected into a cell line expressing HBV genome(HepG2-N10). The cytotoxicity of cells transfected with different expression plasmids were detected by MTT assay. The levels of hepatitis B surface antigen (HBsAg) were determined by using chemiluminescent immunoassay (CLIA). The expression of HBV at mRNA level was analyzed by quantitative real-time PCR (qRT-PCR). The qPCR was performed for the detection of extracellular and intracellular HBV DNA. The next-generation sequencing (NGS) Illumina MiSeq Platform was used to analyze HBV genome editing. Results No significant cytotoxic effects were detected in HepG2-N10 cells transfected with different expression plasmids. Compared with the cells carrying pCas-Guide-GFP-Scramble, the levels of HBsAg in the supernatants of transfected cell culture harboring pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were decreased by 24.2% (P 0.05), respectively. The levels of HBsAg in cells transfected with pCas-Guide-GFP-G1 and pCas-Guide-GFP-G2 were respectively decreased by 16.4% (P>0.05) and 32.1% (P>0.05) as compared with that of pCas-Guide-GFP-Scramble transfected group. The expression of HBV at mRNA level was inhibited as indicated by the results of qRT-PCR. Moreover, the levels of extracellular HBV DNA were respectively suppressed by 23% (P>0.05) and 35% (P 0.05) and 18% (P>0.05). Different types of insertion/deletion mutation were detected in HBV genome by high-throughput sequencing. Conclusion HBV-specific CRISPR/Cas9 system could inhibit the expression of HBV gene and the replication of virus. Therefore, the CRISPR/Cas9 genome editing technology might be used as a potential tool for the treatment of persistent HBV infection. Key words: CRISPR/Cas9 genome editing technology; Hepatitis B virus; Inhibition